Cannabinoid CB1 receptors regulate salivation

Saliva serves multiple important functions within the body that we typically take for granted, such as helping prepare food for swallowing and defense against oral pathogens. Dry mouth is a primary symptom of Sjӧgren’s syndrome and is a side effect of many drug treatments. Cannabis users frequently report dry mouth, but the basis for this is still unknown. If the effects occur via the endogenous cannabinoid signaling system, then this may represent a novel mechanism for the regulation of salivation. We examined expression of cannabinoid CB1 receptors in submandibular salivary gland using immunohistochemistry and tested regulation of salivation by THC and cannabinoid-related ligands. We now report that CB1 receptors are expressed in the axons of cholinergic neurons innervating the submandibular gland. No staining is seen in submandibular gland epithelial cells (acinar and ductal), or myoepithelial cells (MECs). Treatment with THC (4 mg/kg, IP) or the cannabinoid receptor agonist CP55940 (0.5 mg/kg) reduced salivation in both male and female mice 1 h after treatment. CBD had no effect on its own but reversed the effect of THC in a concentration-dependent manner. Neither the CB1 receptor antagonist SR141716 (4 mg/kg) nor the CB2-selective agonist JWH133 (4 mg/kg) had an effect on salivation. We also found that fatty acid amide hydrolase (FAAH), the enzyme that metabolizes the endocannabinoid anandamide and related lipids, regulates salivation. Salivation was reduced in FAAH knockout mice as well as mice treated with the FAAH blocker URB597 (4 mg/kg). URB597 had no effect in CB1 knockout mice. FAAH protein is detected intracellularly in acinar but not ductal epithelial cells. In lipidomics experiments, we found that FAAH knockout mice chiefly had elevated levels of acylethanolamines, including anandamide, and reduced levels of acyglycines. Our results are consistent with a model wherein endocannabinoids activate CB1 receptors on cholinergic axons innervating the submandibular gland. THC likely acts by plugging into this system, activating CB1 receptors to reduce salivation, thus offering a mechanism underlying the dry mouth reported by cannabis users.

Method of measuring salivation. To measure basal salivation in mice we developed a minimally invasive method that makes use of phenol red coated threads (see Fig. 1). Developed initially for ophthalmic use, these threads are discolored by the pH of tears and saliva, which rapidly travel along the thread allowing the distance traveled along the thread to be taken as a measure of tearing/salivation. To allow for consistent placement in the oral cavity, a thread is inserted into a glass capillary (fire-polished to prevent oral injury), leaving 3 mm of thread outside the opening of the capillary. The tip of this glass capillary is then placed under the tongue of an isoflurane anesthetized mouse for 10 s. The induction of anesthesia is rapid (< 3 min) and mice quickly recover from the anesthesia with first signs of recovery ~ 1 min after removal from isoflurane. Animals are ambulatory within ~ 5 min and so spend the bulk of the time between measurements active in their home cages. Drug effects at 1 h are compared to control animals at 1 h using an unpaired t-test unless otherwise stated. For experiments that test the effect of drug treatments, animals are injected with drugs intraperitoneally (IP) 1 h before measurement of salivation. Drug concentrations were determined based on EC50/IC50 values and/or previously reported concentrations in vivo (E.g. Ref. 27 for SR141716).
Immunohistochemistry. For immunohistochemistry, submandibular glands were fixed in 4% paraformaldehyde for 45 min at 4 °C, then placed sequentially in 10% and 30% sucrose in PBS overnight before being suspended in OCT (Thermo Fisher Scientific, Waltham, MA, USA) in a 15 mL plastic test tube, then submerged in cold (− 80 °C) methanol. Our immunohistochemical methods have been published previously (e.g. Ref. 28 ), but briefly: fixed SMG tissue was sectioned on a Leica cryostat (Leica Microsystems, Wetzlar, Germany), then sections were mounted on Superfrost Plus slides (Thermo Fisher). Slides were blocked with BSA, followed by treatment with primary antibodies (in PBS, saponin, 0.2%) for 1-2 days at 4 °C. See Table 1 for list of primary antibodies. In cases where secondary antibodies were required, a second staining with secondary antibody (~ 4 h at RT) was done after washing off the primary antibody. Appropriate secondary antibodies were labeled with the Alexa488, Alexa594, or Alexa647 (Thermo Fisher). Slides were then mounted with mounting media containing 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) to visualize nuclei (Fluoromount, Sigma-Aldrich, St. Louis, MO, USA). Images were acquired with a Leica TCS SP5 (Leica Microsystems). Images were processed using FIJI (vsn 2.3.0/1.53q, available at https:// imagej. net/ Fiji/ downl oads) and/or IMARIS Image Analysis Software (BITPLANE, Oxford Instruments) and/or Photoshop vsn. 21.2 (Adobe Inc., San Jose, CA, USA) software. Images were modified only in terms of brightness and contrast.
Western Blot methods. Salivary glands were harvested and quick frozen on dry ice. Tissue punches (4 mm) were made from the frozen tissue and quick homogenized in RIPA buffer [50 mM Tris, pH8.0, 150 mM   29,30 . Statistical analyses were completed in SPSS Statistics 27 (IBM). One-way ANOVAs followed by Fisher's Least Significant Difference post-hoc analyses were used to determine statistical differences between the average concentration of an analyte measured in WT verses KO SMG. Statistical significance for all tests was set at p < 0.05, and trending significance at 0.05 < p < 0.10. Descriptive and inferential statistics were used to create heatmaps for visualizing changes in lipids 29,30 . Briefly, the direction of changes in the treatment group compared to vehicle are depicted by color, with green representing an increase and orange representing a decrease. Level of significance is shown by color shade, wherein p < 0.05 is a dark shade and 0.05 < p < 0.1 is a light shade. Direction of the change compared to vehicle is represented by up (increase) or down (decrease) arrows. Effect size is represented by the number of arrows, where 1 arrow corresponds to 1-1.49-fold difference, 2 arrows to a 1.5-1.99-fold difference, 3 arrows to a 2-2.99-fold difference, 4 arrows a 3-9.99-fold difference, and 5 arrows a difference of tenfold or more. To calculate the effect size of a lipid that was significantly higher in the treatment group, the average concentration of the drug group is divided by the average concentration of the vehicle group. To calculate the effect size of a compound that is significantly lower in the treatment group, the same calculation is conducted, except the inverse of the resulting ratio is used to represent a fold decrease.

Results
CB1 receptors are expressed in submandibular gland of the mouse.. Using immunohistochemistry, we tested CB1 receptor expression in the submandibular gland (SMG) of the mouse ( Fig. 2A). Specificity of the staining was confirmed in SMG from CB1 knockout mice (CB1 −/− ) (Fig. 2B). We found that myoepithelial and acinar cells had no or little CB1 protein expression, while expression could be seen in axon-like processes ( Fig. 2A,C). Costaining with antibodies against choline acetyl transferase (ChAT), a marker for cholinergic neurons showed frequent co-staining with CB1 ( Fig. 2C-E), suggesting that CB1 is present on parasympathetic afferents.

FAAH protein is expressed in acinar cells of the SMG.
To determine where the FAAH protein is expressed we used immunohistochemistry. FAAH protein has previously been reported to localize to intracellular structures such as smooth endoplasmic reticulum and the membranes of mitochondria 36 . Consistent with this, we found that FAAH protein is expressed intracellularly within acinar cells (Fig. 5A,C-D) but not in ductal cells. Staining is absent in SMG from FAAH knockout mice (Fig. 5B). The actin marker phalloidin labels ducts within acini (e.g. Fig. 5D). FAAH did not appear to exhibit a polarized distribution within acinar cells. We also tested whether FAAH protein levels varied by sex in the submandibular gland using a Western blot assay. We detected a FAAH band at the predicted molecular weight (63kda) and found that levels were trending towards significance with levels higher in females (Fig. 6, p = 0.058 by Student's t-test, n = 4; Full blot in Supplementary  Fig. S1).
We also tested for NAPE-PLD protein expression using a knockout-validated antibody against NAPE-PLD 37 . The most prominent NAPE-PLD protein was seen in a subset of myoepithelial cells (Supplementary Fig S3).

FAAH knockout mice have elevated acyl-ethanolamine levels and decreased acyl-glycine levels.
We examined the lipidomic profile of FAAH knockout mice relative to wild type mice. Previously, we showed the FAAH KO mice had dramatically different lipid profiles in a wide range of lipids in the CNS, though some areas of the CNS were more affected than others with more changes in lipids measured in cortex than   (Fig. 7), we tested the lipidomic profiles of SMG in male WT and FAAH KO mice. We found that levels of the NAEs, AEA, LEA, and DEA were significantly increased; OEA levels were equivalent, and levels of SEA were significantly lower in FAAH KOs. Five species of N-acyl glycines, the 2-AG analog, 2-linoleoyl glycerol, and arachidonoyl taurine were significantly lower.
Blocking MAGL activity does not alter salivation. As noted above, the endocannabinoid 2-AG has also been found to mediate effects of CB1 activation, particularly in the CNS (e.g. Ref. 40 ). Though FAAH appears to account for the CB1-based effects on salivation, we also tested whether blocking MAGL, the chief 2-AG metabolizing enzyme, has an impact on salivation. The MAGL blocker JZL184 (8 mg/kg, IP) did not alter salivation relative to baseline values at one hour after treatment ( Supplementary Fig. S2A, salivation one hour after JZL184 (8 mg/kg) in males: 14.1 ± 0.90, n = 8; in females: 16.2 ± 1.3, n = 8, NS by paired t-test vs. pre-treatment baseline, p = 0.56 for males, p = 0.16 for females). We also tested for MAGL protein expression with an antibody that we have previously used with success in the anterior eye 6 but did not detect MAGL protein expression in submandibular gland ( Supplementary Fig. S2B).

Discussion
Cannabis users frequently report both dry mouth and dry eye as a side-effect. Several studies have examined aspects of how the cannabinoid signaling system might regulate salivation but no study to date has investigated this systematically. This is partly because traditional methods to study salivation in mouse models-and thereby take advantage of cannabinoid-related transgenic models-have, as a rule, involved invasive methods such as cannulation. To facilitate studies of cannabinoid regulation of salivation in mice, we developed a simple noninvasive method to measure basal salivation. Our chief findings are that THC reduces salivation in both males and females in a CB1-dependent manner and that CB1 receptors reside chiefly in cholinergic axons innervating the submandibular gland. We also found evidence that lipid signaling molecules mediate the reduction in salivation since deletion or inhibition of the NAE-metabolizing enzyme FAAH reduces salivation, while FAAH inhibitors had no effect in CB1 knockouts. FAAH protein is expressed within acinar but not ductal epithelial cells in the SMG. In lipidomic findings, FAAH knockout mice have increased levels of NAEs, including anandamide, and decreases in N-acyl glycines, 2-LG and N-arachidonoyl taurine among other changes. A reduction in acetylcholine release by cannabinoids was demonstrated in an elegant study by McConnell et al. 23 showing that THC reduced basal salivary flow and the production of acetylcholine, but did not affect pilocarpine-or acetylcholine-stimulated salivary flow. Our findings indicate that key components of an intact cannabinoid signaling system are present in the submandibular gland. Our results are consistent with the following model of cannabinoid regulation of salivation: CB1 receptors are expressed on the axons of cholinergic neurons that innervate the submandibular gland; these CB1 receptors are activated by endogenous lipids, reducing acetylcholine release and, as a result, basal salivation. Endogenous lipids are then metabolized by FAAH that is expressed in acinar cells (schematized in Fig. 8). In this model, the effects of cannabis on salivation occur via THC activation of the CB1 signaling system in the submandibular gland. We find that CBD alone is without effect on salivation, but that CBD interferes with THC's effects on salivation in a concentration-dependent manner. In www.nature.com/scientificreports/ the context of salivation, CBD reverses the effects of 4 mg/kg of THC with an IC50 of 4.0 mg/kg. This is consistent with reports that CBD antagonizes CB1 receptor activation, likely as a negative allosteric modulator 32,41 . We  e  n  i  r  e  s  l  y  o  t  i  m  l  a  p  -N  e  n  i  n  a  l  a  l  y  o  t  i  m  l  a  p  -N   e  n  i  r  e  s  l  y  o  r  a  e  t  s  -N  e  n  i  n  a  l  a  l  y  o  r  a  e  t  s  -N   e  n  i  r  e  s  l  y  o  e  l  o  -N  e  n  i  n  a  l  a  l  y  o  e  l  o  -N   e  n  i  r  e  s  l  y  o  e  l  o  n  i  l  -N  e  n  i  n  a  l  a  l  y  o  e  l  o  n  www.nature.com/scientificreports/ have found that this has functional physiological consequences in vivo both here and in countering the effects of THC on ocular pressure 9 , but by testing a range of concentrations we have learned that in vivo CBD is roughly equipotent as an inhibitor of THC. In contrast to several published studies, we do not find evidence that CB2 receptor activation alters salivation acutely, i.e. 1 h after treatment. Kopach et al. 24 , Prestifillipo et al. 17 , and Rettori et al. 42 each offered pharmacological evidence for CB2 receptor regulation of salivation. We have previously shown that several nominally selective CB2 agonists/antagonists (JWH15/AM630) also work well at CB1 receptors 43 . Each study also buttressed their findings with immunohistochemical analysis, but none were validated with the use of knockout mice.
Our finding that CB1 protein expression is chiefly neuronal/ axonal in nature contrasts with previous studies that reported CB1 protein in multiple cell types including acini and ductal epithelial cells 17,21,24 . Because those studies made use of rat 16,24 , or piglet 21 , neither was in a position to validate expression patterns in knockout animals, though it is possible that CB1 expression varies by species. Our immunohistochemical findings of CB1 and FAAH expression within the submandibular gland, and NAPE-PLD in myoepithelial cells, suggest the following circuit: NAEs are synthesized locally in myoepithelial cells, then act on CB1 receptors expressed in parasympathetic axons, after which they are broken down locally in non-ductal acinar cells (Fig. 8). The acinar cells would effectively act as a sump for stray/excess NAEs. The absence of an effect of a MAGL blocker on salivation as well as MAGL protein expression using immunohistochemistry is consistent with a primary role for NAEs over 2-AG in this system.
Our study was restricted to basal salivation and submandibular gland expression. Salivation is under complex control, regulated not only by parasympathetic and sympathetic inputs but also by inputs deriving from taste, smell and other sensory modalities. Some of this reflexive salivation occurs through the parotid gland, which adjoins the submandibular gland and has been reported to express functional cannabinoid receptors 20 , but was not the subject of the present study.
In our lipidomic analysis of FAAH KO mice vs. wild type controls, we saw expected increases in N-acyl ethanolamines (NAEs) and declines in N-acyl glycines (NAGlys) but some notable differences relative to findings in other tissues. Interestingly, we found that levels of three NAEs, anandamide, LEA and DHEA, were increased, while the levels of SEA were decreased and only OEA remained unaltered. This suggests that the longer, more unsaturated fatty acid ethanolamines, alone, in combination, or their metabolites, may be more likely to serve as the endogenous messengers mediating the effects of CB1 in the salivary gland. While in the CNS FAAH inhibitors generally raise NAEs as a group 39 , we have shown that this is not always the case in peripheral tissues, such as ileum and colon, where some NAEs either see no changes or even declines with FAAH inhibition 44 . This may be due to lower levels of FAAH expression in some peripheral structures compared to the CNS 45 or www.nature.com/scientificreports/ may be an indication of more complex NAE metabolism in peripheral tissues. As noted above, we also saw substantial reductions in N-acyl glycines in the FAAH KO SMG. This family of lipids appears to require FAAH for synthesis 46 . Broadly speaking, there were fewer overall differences in the SMG lipidome relative to other tissues. For instance we have previously reported changes in N-acyl serines and 2-acyl glyerols in eye of FAAH KO 7 . With a few exceptions such as alterations in N-acyl taurines, the chief consequences of FAAH deletion were changes in NAEs and NAGlys. The role, if any, for these lipids is not always known, but some of these acyl species may be endogenous ligands at other targets. N-arachidonoyl glycine (NAGly), for example, is implicated as an agonist for the cannabinoid-related receptor GPR18 7,47 . One attraction of examining CB1 regulation of both salivation and lacrimation is to allow a comparison across two related exocrine glands. Both tears and saliva are produced by exocrine glands under neuronal control-largely by autonomic inputs-and are partially innervated by the same brain stem nucleus. Given the reports of dry mouth and dry eye by cannabis users, we had predicted that both tearing and salivation would be reduced by CB1 receptor activation independent of sex. We have recently shown in mice that THC regulation of tearing is sex-dependent, with an inhibition in males but an increase in tearing in females 14 . Both effects are dependent on CB1 receptor activation. In contrast, CB1 receptor activation reduces salivation in mice in both sexes, a major difference between these two exocrine glands. The only sex-dependent difference we noted was in the time-course of URB597 effects, with females only reaching a significantly lower salivation at 3 h instead of the 1 h needed with males and a trending elevation in the level of FAAH protein in females. Therefore, while the two exocrine glands see some commonalities, particularly in that CB1 receptors on cholinergic inputs appear to reduce parasympathetic inputs, there are substantial qualitative differences between the two.
In summary, we have examined cannabinoid CB1 receptor regulation of salivation in mice. Though this is not the first study of this topic, this study benefits from the use of transgenic mice, either as gold-standard controls for immunohistochemistry or as controls for functional studies. Therefore, we can propose, with some confidence, a model for CB1 regulation of basal salivation via inhibition of parasympathetic neuronal inputs to the submandibular glands and a role for N-acyl ethanolamines and the metabolic enzyme FAAH. In contrast to our findings for tearing, this regulation of salivation is similar in males and females. Our results also suggest that the 'cottonmouth' frequently reported by cannabis users is due to THC activation of the CB1 receptors and that this is opposed, with similar potency, by CBD. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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